Acetylcholine released from cholinergic neurons in the peripheral and central nervous systems affects many different biological processes through interaction with two major classes of acetylcholine receptors—the nicotinic and the muscarinic acetylcholine receptors. Muscarinic acetylcholine receptors (mAChRs) belong to the superfamily of G-protein coupled receptors having seven transmembrane domains. There are five subtypes of mAChRs, termed M1-M5, and each is the product of a distinct gene. Each of these five subtypes displays unique pharmacological properties. Muscarinic acetylcholine receptors are widely distributed in vertebrate organs, and these receptors can mediate both inhibitory and excitatory actions. For example, in smooth muscle found in the airways, M3 mAChRs mediate contractile responses. For a review, see Caulfield (1993 Pharmac. Ther. 58:319-79).
In the lungs, mAChRs have been localized to smooth muscle in the trachea and bronchi, the submucosal glands, and the parasympathetic ganglia. Muscarinic receptor density is greatest in parasympathetic ganglia and then decreases in density from the submucosal glands to tracheal and then bronchial smooth muscle. Muscarinic receptors are nearly absent from the alveoli. For review of mAChR expression and function in the lungs, please see Fryer and Jacoby (1998 Am J Respir Crit. Care Med 158(5, pt 3) S 154-60).
Three subtypes of mAChRs have been identified as important in the lungs, M1, M2 and M3 mAChRs. The M3 mAChRs, located on airway smooth muscle, mediate muscle contraction. Stimulation of M3 mAChRs activates the enzyme phospholipase C via binding of the stimulatory G protein Gq/11 (Gs), leading to liberation of phosphatidyl inositol-4,5-bisphosphate, resulting in phosphorylation of contractile proteins. M3 mAChRs are also found on pulmonary submucosal glands. Stimulation of this population of M3 mAChRs results in mucus secretion.
M2 mAChRs make up approximately 50-80% of the cholinergic receptor population on airway smooth muscles. Although the precise function is still unknown, they inhibit catecholaminergic relaxation of airway smooth muscle via inhibition of cAMP generation. Neuronal M2 mAChRs are located on postganglionic parasympathetic nerves. Under normal physiologic conditions, neuronal M2 mAChRs provide tight control of acetylcholine release from parasympathetic nerves. Inhibitory M2 mAChRs have also been demonstrated on sympathetic nerves in the lungs of some species. These receptors inhibit release of noradrenaline, thus decreasing sympathetic input to the lungs.
M1 mAChRs are found in the pulmonary parasympathetic ganglia where they function to enhance neurotransmission. These receptors have also been localized to the peripheral lung parenchyma, however their function in the parenchyma is unknown.
Muscarinic acetylcholine receptor dysfunction in the lungs has been noted in a variety of different pathophysiological states. In particular, in asthma and chronic obstructive pulmonary disease (COPD), inflammatory conditions lead to loss of inhibitory M2 muscarinic acetylcholine autoreceptor function on parasympathetic nerves supplying the pulmonary smooth muscle, causing increased acetylcholine release following vagal nerve stimulation (Fryer et al. 1999 Life Sci 64 (6-7) 449-55). This mAChR dysfunction results in airway hyperreactivity and hyperresponsiveness mediated by increased stimulation of M3 mAChRs.
Recent literature has focused on the non-neuronal cholinergic system in the lungs where there is an emerging literature supporting a role for muscarinic receptors in mediating immunomodulatory and inflammatory functions in respiratory diseases such as asthma and COPD. Many of the components for cholinergic signaling have been reported to be contained within inflammatory and resident cells of the lungs, including muscarinic receptor expression on lymphocytes, alveolar macrophages, mast cells and epithelial cells. The view that acetylcholine is solely a neurotransmitter of the parasympathetic nervous system is currently being challenged as there is mounting evidence to suggest it has an integral role in host defense and airway inflammation. For a full review see Gwilt et al., 2007 (Gwilt C R. et al., The non-neuronal cholinergic system in the airways: An unappreciated regulatory role in pulmonary inflammation? Pharmacol. Ther. 2007; 115: 208-222) and Kummer & Lips 2006 (Kummer W and Lips K S. Non-neuronal acetylcholine release and its contribution to COPD pathology. Drug Discovery Today: Disease Mechanisms 2006; 3:47-52). A consequence of this emerging science is the implication that anti-cholinergic antagonists may have a much broader therapeutic potential for respiratory diseases with anti-inflammatory and disease modifying activity as well as the their well established utility as bronchodilator agents.
COPD is an imprecise term that encompasses a variety of progressive health problems including chronic bronchitis, chronic bronchiolitis and emphysema, and it is a major cause of mortality and morbidity in the world. Smoking is the major risk factor for the development of COPD; nearly 50 million people in the U.S. alone smoke cigarettes, and an estimated 3,000 people take up the habit daily. As a result, COPD is expected to rank among the top five as a world-wide health burden by the year 2020. Inhaled anti-cholinergic therapy is currently considered the “gold standard” as first line therapy for COPD (Pauwels et al. 2001 Am. J. Respir. Crit. Care Med. 163:1256-1276).
Despite the large body of evidence supporting the use of anti-cholinergic therapy for the treatment of airway hyperreactive diseases, relatively few anti-cholinergic compounds are available for use in the clinic for pulmonary indications. Ipratropium Bromide (Atrovent©; and Combivent©, in combination with albuterol) is one of the few inhaled anti-cholinergic marketed for the treatment of airway hyperreactive diseases. While this compound is a potent anti-muscarinic agent, it is short acting, and thus must be administered as many as four times daily in order to provide relief for the COPD patient. The long-acting anti-cholinergic Tiotropium Bromide (Spiriva©) has recently been approved in a number of countries.
Since mAChRs are widely distributed throughout the body, the ability to apply anti-cholinergics locally and/or topically to the respiratory tract is particularly advantageous, as it would allow for lower doses of the drug to be utilized. Furthermore, the ability to design topically active drugs that have long duration of action, and in particular, are retained either at the receptor or by the lung, would allow the avoidance of unwanted side effects that may be seen with systemic anti-cholinergic use.
WO 2004/091482 describes a dimeric bicyclic amine derivative having anti-muscarinic receptor activity:
wherein, inter alia, X represents a group of the formula (d) or (e):—Y—Ar—Y—  (d)—Y-L-Y—  (e)
Y is selected from the group consisting of a bond, OR2, SR2, NR2R3, and C1-4 alkyl; and L represents a bond, C1-4 alkyl or C3-8 cycloalkyl.
WO 2005/095407 also discloses a similar dimeric bicyclic amine derivative to that above having anti-muscarinic receptor activity wherein inter alia, X is a group of the formula (d), (e) and (f):—Y—Ar—Y—  (d)—Y-L-Y—  (e)Y—Ar1—Z—Ar2—Y  (f)
Y is, independently, selected from the group consisting of a bond, O, S, NR2, —NR2C1-4 alkyl-, and C1-4 alkyl-; each of the alkyl groups may contain a heteroatom selected from O, NR2, or S; and
Z represents a bond, O, NR2, S, C1-4 alkylidene or C1-4 alkyl.
Other mAChR antagonists, non-dimeric in structure, may be found in WO 2004/012684; WO 2005/009439; WO 2005/09362; WO 2005/09440; WO 2005/037280; WO 2005/037224; WO 2005/046586; WO 2005/055940; WO 2005/055941; WO 2005/067537; WO 2005/087236; WO 2005/086873; WO 2005/094835; WO 2005/094834; WO 2005/094251; WO 2005/099706; WO 2005/104745; WO 2005/112644; WO 2005/118594; WO 2006/005057; WO 2006/017767; WO 2006/017768; WO 2006/050239; WO 2006/055503; WO 2006/055553; WO 2006/062931; WO 2006/062883; WO 2006/065788; WO 2006/065755; WO 2007/018514; WO 2007/018508; WO 2007/016639; WO 2007/016650; and WO 2007/022351.
NVA237 (glycopyrrolate) glycopyrrolate or glycopyrronium bromide, a quaternary ammonium derivative with anticholinergic and antimuscarinic properties. It is being developed by Novartis for once daily treatment of COPD.

LAS-34273, also known as aclidinium bromide, is a quaternary ammonium anticholinergic muscarinic M3 antagonist originated by Almirall and believed to be in phase 3 development for treating COPD.

With respect to the PDE4 moieties: U.S. Pat. No. 3,979,399, U.S. Pat. No. 3,840,546, and U.S. Pat. No. 3,966,746 (E. R. Squibb & Sons) disclose 4-amino derivatives of pyrazolo[3,4-b]pyridine-5-carboxamides wherein the 4-amino group NR3R4 can be an acyclic amino group wherein R3 and R4 may each be hydrogen, lower alkyl (e.g. butyl), phenyl, etc.; NR3R4 can alternatively be a 3-6-membered heterocyclic group such as pyrrolidino, piperidino and piperazino. The compounds are disclosed as central nervous system depressants useful as ataractic, analgesic and hypotensive agents.
U.S. Pat. No. 3,925,388, U.S. Pat. No. 3,856,799, U.S. Pat. No. 3,833,594 and U.S. Pat. No. 3,755,340 (E. R. Squibb & Sons) disclose 4-amino derivatives of pyrazolo[3,4-b]pyridine-5-carboxylic acids and esters. The compounds are mentioned as being central nervous system depressants useful as ataractic agents or tranquilizers, as having anti-inflammatory and analgesic properties. The compounds are mentioned as increasing the intracellular concentration of adenosine-3′,5′-cyclic monophosphate and for alleviating the symptoms of asthma.
H. Hoehn et al., J. Heterocycl. Chem., 1972, 9(2), 235-253 discloses a series of 1H-pyrazolo[3,4-b]pyridine-5-carboxylic acid derivatives with 4-hydroxy, 4-chloro, 4-alkoxy, 4-hydrazino, and 4-amino substituents. Ethyl 4-(n-butylamino)-1-ethyl-1H-pyrazolo[3,4-b]-pyridine-5-carboxylate is disclosed therein; this compound is cartazolate.
The compound tracazolate, ethyl 4-(n-butylamino)-1-ethyl-6-methyl-1H-pyrazolo[3,4-b]-pyridine-5-carboxylate, is known as an anxiolytic agent (e.g. see J. B. Patel et al., Eur. J. Pharmacol, 1982, 78, 323). Other 1-substituted 4-(NH2 or NH-alkyl)-1H-pyrazolo[3,4-b]-pyridine-5-carboxylic acid esters and amides are disclosed as potential anxiolytic agents in T. M. Bare et al., J. Med. Chem., 1989, 32, 2561-2573.
CA 1003419, CH 553 799 and T. Denzel, Archiv der Pharmazie, 1974, 307(3), 177-186 disclose 4,5-disubstituted 1H-pyrazolo[3,4-b]pyridines unsubstituted at the 1-position.
Japanese laid-open patent application JP-2002-20386-A (Ono Yakuhin Kogyo KK) published on 23 Jan. 2002 discloses pyrazolopyridine compounds of the following inter alia formula:

The compounds of JP-2002-20386-A are stated as having PDE4 inhibitory activity and as being useful in the prevention and/or treatment of inflammatory diseases and many other diseases.
1,3-Dimethyl-4-(arylamino)-pyrazolo[3,4-b]pyridines with a 5-C(O)NH2 substituent similar or identical to those in JP-2002-20386-A were disclosed as orally active PDE4 inhibitors by authors from Ono Pharmaceutical Co. in: H. Ochiai et al., Bioorg. Med. Chem. Lett., 2004, vol. 14(1), pp. 29-32. Full papers on these and similar compounds as orally active PDE4 inhibitors are: H. Ochiai et al., Bioorg. Med. Chem., 2004, 12(15), 4089-4100, and H. Ochiai et al., Chem. Pharm. Bull, 2004, 52(9), 1098-1104.
EP 0 076 035 A1 (ICI Americas) discloses pyrazolo[3,4-b]pyridine derivatives as central nervous system depressants useful as tranquilizers or ataractic agents for the relief of anxiety and tension states.
J. W. Daly et al., Med. Chem. Res., 1994, 4, 293-306 and D. Shi et al., Drug Development Research, 1997, 42, 41-56 disclose a series of 4-(amino)substituted 1H-pyrazolo[3,4-b]pyridine-5-carboxylic acid derivatives, including ethyl 4-cyclopentylamino-1-methyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate, and their affinities and antagonist activities at A1- and A2A-adenosine receptors, and the latter paper discloses their affinities at various binding sites of the GABAA-receptor channel. S. Schenone et al., Bioorg. Med. Chem. Lett., 2001, 11, 2529-2531, and F. Bondavalli et al., J. Med. Chem., 2002, 45(22), pp. 4875-4887 disclose a series of 4-amino-1-(2-chloro-2-phenylethyl)-1H-pyrazolo[3,4-b]pyridine-5-carboxylic acid ethyl esters as A1-adenosine receptor ligands.
WO 02/060900 A2 appears to disclose, as MCP-1 antagonists for treatment of allergic, inflammatory or autoimmune disorders or diseases, a series of bicyclic heterocyclic compounds with a —C(O)—NR4—C(O)—NR5R6 substituent, including isoxazolo[5,4-b]pyridines and 1H-pyrazolo[3,4-b]pyridines (named as pyrazolo[5,4-b]pyridines) with the —C(O)—NR4—C(O)—NR5R6 group as the 5-substituent and optionally substituted at the 1-, 3-, 4-, and/or 6-positions. Bicyclic heterocyclic compounds with a —C(O)NH2 substituent instead of the —C(O)—NR4—C(O)—NR5R6 substituent are alleged to be disclosed in WO 02/060900 as intermediates in the synthesis of the —C(O)—NR4—C(O)—NR5R6 substituted compounds. See also WO 02/081463 A1 for similar MCP-1 antagonists.
WO 00/15222 (Bristol-Myers Squibb) discloses inter alia pyrazolo[3,4-b]pyridines having inter alia a C(O)—X1 group at the 5-position and a group E1 at the 4-position of the ring system. Amongst other things, X1 can for example be —OR9, —N(R9)(R10) or —N(R5)(-A2-R2), and E1 can for example be —NH-A1-cycloalkyl, —NH-A1-substituted cycloalkyl, or —NH-A1-heterocyclo; wherein A1 is an alkylene or substituted alkylene bridge of 1 to 10 carbons and A2 can for example be a direct bond or an alkylene or substituted alkylene bridge of 1 to 10 carbons. The compounds are disclosed as being useful as inhibitors of cGMP phosphodiesterase, especially PDE type V, and in the treatment of various cGMP-associated conditions such as erectile dysfunction.
H. de Mello, A. Echevarria, et al., J. Med. Chem., 2004, 47(22), 5427-5432, discloses 3-methyl or 3-phenyl 4-anilino-1H-pyrazolo[3,4-b]pyridine 5-carboxylic esters as potential anti-Leishmania drugs.
WO 2004/056823 A1 (PCT/EP2003/014867, filed on 19 Dec. 2003, published on 8 Jul. 2004, Glaxo Group Limited), and incorporated herein by reference in its entirety as though fully set forth, discloses and claims pyrazolo[3,4-b]pyridine compounds or salts thereof with a 4-NR3R3a group (R3a is preferably H) and with a group Het at the 5-position of the pyrazolo[3,4-b]pyridine, wherein Het is usually a 5-membered optionally substituted heteroaryl group.
WO 2004/056823 A1 also discloses the use of these compounds as PDE4 inhibitors and for the treatment and/or prophylaxis of inter alia COPD, asthma or allergic rhinitis.
WO 2004/024728 A2 (PCT/EP2003/011814, filed on 12 Sep. 2003, published on 25 Mar. 2004, Glaxo Group Limited), discloses pyrazolo[3,4-b]pyridine having the following generic formula.

In WO 2004/024728 A2, pyrazolo[3,4-b]pyridine compounds are disclosed as being inhibitors of PDE4. WO 2004/024728 and WO 2004/056823 are noted in Expert Opin. Ther. Patents, 2005 (January edition), 15(1), 111-114.
WO 2005/058892 A1 (PCT/EP2004/014490, filed on 17 Dec. 2004, published on 30 Jun. 2005, Glaxo Group Limited), discloses pyrazolo[3,4-b]pyridine compounds for use as PDE4 inhibitors for treating inflammatory or allergic diseases such as COPD, asthma, rheumatoid arthritis, allergic rhinitis or atopic dermatitis.
Further pyrazolo[3,4-b]pyridine compounds and their use as PDE4 inhibitors, are disclosed in patent applications WO 2005/090353 A1 (PCT/GB2005/000976), WO 2005/090348 A1 (PCT/GB2005/000983), WO 2005/090354 A1 (PCT/GB2005/000987), and WO 2005/090352 A1 (PCT/EP2005/003038) (all Glaxo Group Limited). PCT/EP2005/003038, PCT/GB2005/000987 and PCT/GB2005/000983, were all filed 15 Mar. 2005.
WO 03/087064 is directed to compounds having both antagonism of the M3 muscarinic receptor and inhibition of PDE4, having the formula:
wherein, inter alia, Y is —NH—R2 or
Two subsequent papers describe in vitro profiles of the lead compounds and in vivo activity after intranasal dosing. Provins, L., et al., Bioorganic & Medicinal Chemistry Letters, 16: 1834-1839 (2006), and Provins, L. et al., Bioorganic & Medicinal Chemistry Letters, 17:3007-3080 (2007). Although promising the data demonstrates that the compounds do not display the in vitro profile that will deliver an in vivo profile displayed by compounds optimized for each molecular target.
Therefore, there is still a need for compounds which contain both the strength and benefit of a combination of PDE4 inhibitory activity and the muscarinic antagonist activity for the treatment of and/or prophylaxis of respiratory diseases, such as chronic obstructive pulmonary disease (COPD), asthma, or inflammatory or allergic diseases such as rhinitis (e.g. allergic rhinitis), atopic dermatitis or psoriasis. The present invention is directed to the novel concept of providing a dual pharmacophore which has both activities.